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Forest ecosystems with low soil nitrogen (N) availability are characterized by direct competition for this growth-limiting resource between several players, i.e. various components of vegetation, such as old-growth trees, natural regeneration and understorey species, mycorrhizal fungi, free-living fungi and bacteria. With the increase in frequency and intensity of extreme climate events predicted in current climate change scenarios, also competition for N between plants and/or soil microorganisms will be affected. In this review, we summarize the present understanding of ecosystem N cycling in N-limited forests and its interaction with extreme climate events, such as heat, drought and flooding. More specifically, the impacts of environmental stresses on microbial release and consumption of bioavailable N, N uptake and competition between plants, as well as plant and microbial uptake are presented. Furthermore, the consequences of drying-wetting cycles on N cycling are discussed. Additionally, we highlight the current methodological difficulties that limit present understanding of N cycling in forest ecosystems and the need for interdisciplinary studies.

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Long-term nitrogen deposition into forest ecosystems has turned many forests in Central Europe and North America from N-limited to N-saturated systems, with consequences for climate as well as air and groundwater quality. However, complete quantification of processes that convert the N deposited and contributed to ecosystem N cycling is scarce. In this study, we provide the first complete quantification of external and internal N fluxes in an old-growth spruce forest, the Höglwald, Bavaria, Germany, exposed to high chronic N deposition. In this forest, N cycling is dominated by high rates of mineralisation of soil organic matter, nitrification and immobilisation of ammonium and nitrate into microbial biomass. The amount of ammonium available is sufficient to cover the entire N demand of the spruce trees. The data demonstrate the existence of a highly dynamic internal N cycle within the soil, driven by growth and death of the microbial biomass, which turns over approximately seven times each year. Although input and output fluxes are of high environmental significance, they are low compared to the internal fluxes mediated by microbial activity.

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AIMS: To study the effect of pH, temperature and substrate on the magnitude of N(2)O and NO production by heterotrophic nitrifiers. METHODS AND RESULTS: The change in N(2)O and NO production by the heterotrophic nitrifiers Alcaligenes faecalis subsp. parafaecalis and Paracoccus pantotrophus because of variations in pH, temperature and substrate was studied in chemostat cultures under steady-state conditions. N(2)O, NO and CO(2) production increased with temperature between 4 and 32 degrees C. For N(2)O an optimum temperature of 28 degrees C was observed. No optimum temperature was found for NO. Highest N(2)O and CO(2) productions were observed at a pH of 7.0. However, besides having an optimum at pH 7.0, especially NO production but also N(2)O production increased significantly at pH

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A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the alpha-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils.

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The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N2O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized gamma-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C2H2, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers.

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Measurements of methane emission rates and concentrations in the soil were made during four growing seasons at the International Rice Research Institute in the Philippines, on plots receiving different levels of organic input. Fluxes were measured using the automated closed chambers system (total emission) and small chambers installed between plants (water surface flux). Concentrations of methane in the soil were measured by collecting soil cores including the gas phase (soil-entrapped methane) and by sampling soil solution in situ (dissolved methane). There was much variability between seasons, but total fluxes from plots receiving high organic inputs (16-24 g CH4 m(-2)) always exceeded those from the low input plots (3-9 g CH4 m(-2)). The fraction of the total emission emerging from the surface water (presumably dominated by ebullition) was greater during the first part of the season, and greater from the high organic input plots (35-62%) than from the low input plots (15-23%). Concentrations of dissolved and entrapped methane in the low organic input plots increased gradually throughout the season; in the high input plots there was an early-season peak which was also seen in emissions. On both treatments, periods of high methane concentrations in the soil coincided with high rates of water surface flux whereas low concentrations of methane were generally associated with low flux rates.

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Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.

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We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.

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In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.

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The present communication describes the properties of isocitrate dehydrogenase in crude extracts from the unicellular Anacystis nidulans and from heterocysts and vegetative cells of Nostoc muscorum and Anabaena cylindrica. The activity levels of this enzyme are much higher in heterocysts than in vegetative cells of N. muscorum and A. cylindrica. Isocitrate dehydrogenase is virtually inactive in vegetative cells of A. cylindrica. The enzyme is negatively regulated by the reduction charge and scarcely affected by oxoglutarate in the three cyanobacteria. The inhibition by ATP and ADP is competitive with respect to isocitrate and NADP+ in A. cylindrica and N. muscorum and noncompetitive in A. nidulans. Isocitrate dehydrogenase from the three cyanobacteria seems to be a hysteretic enzyme. All the experimental data suggest that the major physiological role of isocitrate and the isocitrate dehydrogenase in heterocysts is not to generate reducing equivalents for N2-fixation. Oxoglutarate formed by the enzyme reaction is likely required for the biosynthesis of glutamate inside the heterocysts. Thioredoxin preparations from spinach chloroplasts or from A. cylindrica activate isocitrate dehydrogenase from either heterocysts or vegetative cells of A. cylindrica. Activation is completed within seconds and requires dithiothreitol besides thioredoxin. The thioredoxin preparation which activates isocitrate dehydrogenase also activates NADP+-dependent malate dehydrogenase from spinach chloroplasts or heterocysts of A. cylindrica. Isocitrate dehydrogenase from A. cylindrica is deactivated by oxidized glutathione. It is speculated that isocitrate dehydrogenase and thioredoxin play a role in the differentiation of vegetative cells to heterocysts.

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